PROJECT SUMMARY The long-term objective of this research is to use vibrational reporters and infrared (IR) spectroscopy to study protein and nucleic acid hydration, structure, and dynamics. Nitriles (CN), azides (N3), and selenocyanates (SeCN) are sensitive, site-specific, and relatively non- invasive vibrational reporters with sufficient spatial and temporal resolution. Vibrational coupling between pairs of reporters in the same biomolecule permits the distance and angle between them to be determined, especially using two-dimensional IR spectroscopy. These reporters will be utilized to investigate local biomolecular environments in serine proteases (subtilisin and trypsin), adenylate kinase (ADK), superfolder green fluorescent protein (sfGFP), and RNA tetraloops. Serine proteases are the prototypical model of enzymatic rate enhancements and convergent evolution. They have also been shown to play essential roles in important biomedical processes such as cell differentiation, blood coagulation, apoptosis, and immunity. Mutations in ADK are associated with hemolytic anemia, reticular dysgenesis, and ciliary dyskinesia and ADK6 has been implicated in colorectal cancer. RNA structure is complex and the information gained from this research is expected to benefit future advances in CRISPR and RNA interference (RNAi) therapies. Understanding RNA structure is also critical as one way the innate human immune system recognizes pathogens, such as viruses, is through their RNA profiles. Linear (1D) and/or two-dimensional (2D) IR spectroscopy will be used to study protease active sites with sulfonate inhibitors containing vibrational reporters, ADK with genetically incorporated vibrational reporter unnatural amino acids, and RNA tetraloops with synthetically incorporated nucleotides containing vibrational reporters. Protein X-ray crystallography in collaboration with Christine Phillips-Piro (F&M) will provide additional structural data to complement the IR spectroscopic investigations. Vibrational distance reporter pairs will be used to determine molecular distances between reporters incorporated into sfGFP, ADK, and an RNA hairpin tetraloop in collaboration with Matthew Tucker (U. Nevada, Reno) based upon the strength of vibrational coupling between the reporters. Franklin & Marshall College undergraduate students will play an integral role this biomedical health-related research.